ISOLATION AND CHARACTERIZATION OF FIBROUS SHEATH OF RAT EPIDIDYMAL SPERMATOZOA
ISOLATION AND CHARACTERIZATION OF FIBROUS SHEATH OF RAT EPIDIDYMAL SPERMATOZOA
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DOI：
10.1095/biolreprod14.5.517
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发表时间：
1976-01-01
影响因子：
3.6
通讯作者：
FAWCETT, DW
中科院分区：
文献类型：
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作者：
OLSON, GE;HAMILTON, DW;FAWCETT, DW
从大鼠附睾头和附睾尾精子中分离出纤维鞘，并测定其多肽组成和亚基亚基结构。用Triton X-100-二硫苏糖醇(DTT)、硫氰酸钾(KSCN)DTT和尿素-DTT依次提取附睾尾精子，用电子显微镜检测特定鞭毛成分的损失与用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定特定多肽成分的增溶相关。Triton X-100处理对所有鞭毛膜都有增溶作用，但对鞭毛结构元素没有影响。KSCN提取影响了9+2轴丝的组织，溶解了中央的一对微管和部分b亚管，并破坏了剩余的轴丝基质成分。KSCN也破坏了外层致密纤维皮质，但没有将其移除。十二烷基硫酸钠凝胶显示，KSCN提取的Triton提取的精子中没有一条主要多肽带被去除。短暂的尿素提取导致了特定的外层致密纤维多肽的增溶；外层致密纤维的未溶解部分呈现出非常规则的开塞螺旋构型。长时间的尿素提取完全溶解了外部致密的纤维和连接件，留下纤维鞘作为唯一完整的鞭毛细胞器。用Triton X-100和十二烷基硫酸钠连续示踪附睾头精子，差速离心法提取附睾头精子的纤维鞘。SDS-聚丙烯酰胺凝胶电泳法显示，大鼠精子纤维鞘主要由一种相对分子质量为80000道尔顿的多肽组成。通过对去膜精子的负染对纤维鞘亚结构的研究表明，肋骨由平行的5-6 nm宽的细丝组成，看起来有串珠的亚结构。
The fibrous sheath was isolated from epididymal spermatozoa of rat caput epididymidis and cauda epididymidis and its polypeptide composition and subunit substructure determined. Cauda epididymal spermatozoa were extracted sequentially with Triton X-100-dithiothreitol (DTT), potassium thiocyanate (KSCN) DTT and urea-DTT; the loss of specific flagellar components as determined by EM was correlated with the solubilization of specific polypeptide components as determined by SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis. Triton X-100 treatment solubilized all flagellar membranes but had no effect on the flagellar structural elements. KSCN extraction affected the organization of the 9 + 2 axoneme, solubilizing the central pair of microtubules, portions of the b-subtubule and disorganizing the remaining axoneme matrix components. KSCN also disorganized the outer dense fiber cortex but did not remove it. SDS gels show that none of the major polypeptide bands present in Triton extracted sperm were removed by KSCN extraction. Brief urea extraction resulted in solubilization of specific outer dense fiber polypeptides; the nonsolubilized portion of the outer dense fiber assumed a very regular corkscrew configuration. Prolonged urea extraction completely solubilized the outer dense fibers and connecting piece, leaving the fibrous sheath as the only intact flagellar organelle. Caput epididymal spermatozoa were traced sequentially with Triton X-100 and SDS and the fibrous sheath was recovered by differential centrifugation. SDS-polyacrylamide gel electrophoresis showed that the fibrous sheath of rat sperm is composed predominantly of a single polypeptide with a MW of 80,000 daltons. Study of the substructure of the fibrous sheath, by negative staining of demembranated spermatozoa, show that the ribs are composed of parallel 5-6 nm wide filaments which appear to have a beaded substructure.